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TECHNOLOGY DEVELOPMENT |
aGlykos Finland Ltd., Helsinki, Finland;
bFinnish Red Cross Blood Service, Helsinki, Finland;
cBiomedicum Helsinki, University of Helsinki, Finland;
dFamily Federation of Finland, Infertility Clinic, Helsinki, Finland;
eInstitute of Biomedicine, Department of Medical Chemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden;
fDepartment of Surgery, Clinical Research Center, University of Oulu, Oulu, Finland;
gHospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland
Key Words. Stem cell • Xenoantigen • N-Glycolylneuraminic acid • Clinical trial
Correspondence: Jarmo Laine, M.D., Ph.D., Department of Research and Development, Finnish Red Cross Blood Service, Kivihaantie 7, 00310 Helsinki, Finland. Telephone: +358 9 580 1583; Fax: +358 9 580 1233; e-mail: jarmo.laine{at}veripalvelu.fi
Received on July 19, 2006;
accepted for publication on September 19, 2006.
First published online in STEM CELLS EXPRESS September 28, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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It was recently reported that hESC lines grown in contact with mouse feeder cells and animal-derived cell culture medium components are contaminated with the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) [6]. The authors also suggested that the immunogenic contamination would be difficult to completely eliminate from cell lines and thus negatively influence their use for human therapy. Since almost all currently existing hESC lines have been derived under conditions where they have become exposed to animal material, this would represent a major setback for the development of cellular therapies based on hESCs. The extent of the potential contamination has remained unclear, because it is not known whether similar contamination would afflict other stem cell types or hESCs cultured on human feeder cells.
We have undertaken a project of glycome analysis of various human stem cell types, during which we encountered several Neu5Gc-containing glycoconjugates in stem cells. In the present work, in vitro-cultured human hESC and MSC lines were investigated to study the Neu5Gc contamination at the molecular level, identify sources of contamination, and establish whether the contamination would be reversible in stem cell progeny.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Bone-Marrow Derived Mesenchymal Stem Cell Lines
Bone marrow (BM)-derived MSCs were obtained as described previously [10]. In brief, bone marrow obtained during orthopedic surgery was cultured in minimum essential 
-medium (-MEM), supplemented with 20 mM HEPES, 10% FBS, 1x penicillin-streptomycin, and 2 mM L-glutamine (all from Gibco). After a cell attachment period of 2 days, the cells were washed with Ca2+ and Mg2+-free phosphate-buffered saline (PBS) (Gibco), subcultured further by plating the cells at a density of 2,000 to 3,000 cells/cm2 in the same media and removing half of the media and replacing it with fresh media twice a week until near confluence. For analysis of the effects of human serum on Neu5Gc decontamination, the cells were cultured for 3 weeks in the media, where FBS was replaced by 10% heat-inactivated blood group AB Rh-negative human serum from a male donor.
Flow Cytometric Analysis of Mesenchymal Stem Cell Phenotype
BM MSCs were phenotyped by flow cytometry (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against CD13, CD14, CD29, CD34, CD44, CD45, CD49e, CD73, and HLA-ABC (all from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), CD105 (Abcam Ltd., Cambridge, U.K., http://www.abcam.com), and CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) were used for direct labeling. Appropriate FITC- and PE-conjugated isotypic controls (BD Biosciences) were used. Unconjugated antibodies against CD90 and human leukocyte antigens (HLA)-DR (both from BD Biosciences) were used for indirect labeling. For indirect labeling, FITC-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich) was used as a secondary antibody. The cells were negative for CD14, CD34, CD45, and HLA-DR and positive for CD13, CD29, CD44, CD90, CD105, and HLA-ABC.
Osteogenic Differentiation of Mesenchymal Stem Cells
To induce the osteogenic differentiation of the BM-derived MSCs, the cells were seeded in their normal proliferation medium at a density of 3 x 103/cm2 on 24-well plates (Nalge Nunc, Roskilde, Denmark, http://www.nalgenunc.com/). The next day, the medium was changed to osteogenic induction medium, which consisted of -MEM (Gibco) supplemented with 10% FBS (Gibco), 0.1 µM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.nuncbrand.com), 10 mM ß-glycerophosphate, 0.05 mM L-ascorbic acid-2-phosphate (Sigma-Aldrich), and penicillin-streptomycin (Gibco). The cells were cultured for 3 weeks, changing the medium twice a week before preparing samples for glycome analysis.
Cell Harvesting for N-Glycome Analysis
One milliliter of cell culture medium was saved for N-glycome analysis, and the rest of the medium was removed by aspiration. Cell culture plates were washed with PBS buffer, pH 7.2. PBS was aspirated and cells scraped and collected with 5 ml of PBS (repeated two times). At this point, a small cell fraction (10 µl) was taken for cell counting and the rest of the sample centrifuged for 5 minutes at 400g. The supernatant was aspirated and the pellet washed in PBS an additional two times. The cells were collected with 1.5 ml of PBS, transferred from a 50-ml tube into a 1.5-ml collection tube, and centrifuged for 7 minutes at 5,400 rpm. The supernatant was aspirated, and washing was repeated one more time. The cell pellet was stored at –70°C and used for N-glycome analysis.
Glycan Isolation
Asparagine-linked glycans were detached from cellular glycoproteins by F. meningosepticum N-glycosidase F digestion (Calbiochem, San Diego, http://www.emdbiosciences.com), essentially as described [11]. The released asparagine-linked glycans (N-glycans) were purified for analysis mainly by miniaturized solid-phase extraction steps as described (T. Satomaa, A. Helskanen, M. Mikkola, C. Olsson, M. Blomqvist, T. Jaatinen, O. Aitio, A. Olonen, J. Helin, J. Natunen, T. Tuuri, T. Otankaski, J. Saarinen, J. Laine, unpublished observations).
Mass Spectrometry
Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry was performed with a Bruker Ultraflex TOF/TOF instrument (Bruker, Rheinstetten, Germany, http://www.bruker.de/) essentially as described [12]. Relative molar abundancies of neutral and sialylated N-glycan components can be assigned based on their relative signal intensities in the mass spectra [13]. The mass spectrometric fragmentation analysis was done according to manufacturer's instructions.
Neu5Gc-Specific Antibody Procedures
The Neu5Gc-specific monoclonal antibody P3Q against the glycolipid antigen GM3(Gc) was a kind gift from Dr. Ana Maria Vázquez and Dr. Ernesto Moreno from the Center of Molecular Immunology, Havana, Cuba. It is essentially similar in binding specificity as described previously [7, 8] and was characterized to have binding specificity also against the Neu5Gc3nLc4-Cer glycolipid structure by binding to glycolipid standard molecules on thin-layer chromatography plates. The P3Q antibody was used for detection of N-glycolylneuraminic acid-containing fractions according to the following procedure. Mixtures of glycolipids were separated on thin layer chromatography plates, after which the plates were covered with a plastic layer as described earlier [14]. The plates were then incubated in 2% bovine serum albumin (BSA) in PBS for 1 hour and overlaid with the anti-N-glycolyl antibody diluted with 2% BSA in PBS. After 2 hours, the plates were washed with PBS and overlaid with a second antibody, horseradish peroxidase-labeled antibody (rabbit anti-human IgG 
chains, P.0214; DAKO, Glostrup, Denmark, http://www.dako.com), diluted with 2% BSA in PBS. After an additional 2 hours, the plates were washed with PBS, and the binding fractions were visualized using 0.02% 3,3'-diaminobenzidine tetrahydrochloride in PBS containing 0.03% H2O2. All steps were performed at room temperature. The P3 antibody was confirmed to bind strongly to N-glycolyl-containing glycolipids and, in higher concentrations, also to sulfated glycolipids.
Immunohistochemistry
BM MSCs on passages 9 to 14 were grown on 0.01% poly-L-lysine (Sigma-Aldrich)-coated glass eight-chamber slides (Lab-TekII; Nalge Nunc) at 37°C with 5% CO2 for 2–4 days. After culturing, cells were rinsed five times with PBS (10 mM sodium phosphate, pH 7.2, 140 mM NaCl) and fixed with 4% PBS-buffered paraformaldehyde, pH 7.2, at room temperature (RT) for 10 minutes, followed by washings three times 5 minutes with PBS. Some MSC samples were extracted with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes at RT before blocking. In addition, specificity of the staining was confirmed by sialidase treatment after fixation. In brief, fixed MSC samples were incubated with 10 mU of sialidase (Arthrobacter ureafaciens; Glyko, San Leandro, CA, http://www.prozyme.com) in 50 mM sodium acetate buffer, pH 5.5, overnight at 37°C. After detergent extraction or sialidase treatment, cells were washed three times with PBS. After fixation and different treatments, the nonspecific binding sites were blocked with 3% human serum albumin (HAS)-PBS (Finnish Red Cross Blood Service, Helsinki, Finland, http://www.redcross.fi/en_GB/etusivu/) for 30 minutes at RT. Primary antibodies were diluted in 1% HSA-PBS and incubated for 60 minutes at RT, followed by washing three times for 10 minutes with PBS. Secondary antibody, FITC-labeled goat-anti-mouse (Sigma-Aldrich), was diluted 1:300 in 1% HSA-PBS and incubated for 60 minutes at RT in the dark. Furthermore, cells were washed three times for 5–10 minutes with PBS and mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) stain (Vector Laboratories, Peterborough, U.K., http://www.vectorlabs.com/). Immunostainings were analyzed with Zeiss Axioskop 2-plus fluorescence microscope (Carl Zeiss Vision GmbH, Jena, Germany, http://www.zeiss.com) with FITC and DAPI filters. Images were taken with a Zeiss AxioCam MRc camera and with AxioVision Software 3.1/4.0 (Carl Zeiss) with the 400x magnification.
Biological Reagents
Bovine serum apotransferrin and fetuin were from Sigma-Aldrich.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Immunohistochemical Detection of Neu5Gc in Stem Cells
To verify the results obtained by mass spectrometry, immunohistochemical analyses using a Neu5Gc-specific monoclonal antibody [7, 8] against the glycolipid antigens Neu5Gc3LacCer (GM3Gc) and Neu5Gc3nLc4Cer were performed (Fig. 2). Glycans containing Neu5Gc were detected on both bone marrow-derived MSCs (Fig. 2A–2D) and hESCs (Fig. 2E). Cell permeabilization and extraction with detergent at room temperature abolished the staining, suggesting antigen localization in detergent-soluble areas on the cell surface (Fig. 2B). The results suggest that in addition to glycoprotein glycans, Neu5Gc contamination can also be present in lipid-linked glycans in stem cells.
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-MEM) supplemented with FBS [10]. In addition, the MSC medium contained Neu5Gc structures (Fig. 1E). However, the relative Neu5Gc levels of N-glycans in FBS were lower than in serum replacement medium or in bovine transferrin. Furthermore, a glycoprotein component of FBS, fetuin (Fig. 1F), contained considerably less Neu5Gc than transferrin, suggesting that Neu5Gc contents of animal-derived proteins vary even within an animal species similarly as previously reported [15]. Taken together, all the media used in stem cell culture contained Neu5Gc, but there were significant differences in the relative concentrations. Significantly, the commercial serum replacement medium proved to be a more potent source of Neu5Gc than FBS. The relative Neu5Gc contents in the culture media correlated with the observed high Neu5Gc levels in hESCs (Fig. 1A) and significantly lower Neu5Gc levels in MSCs (Fig. 1B).
Neu5Gc in Stem Cell Progeny
To determine whether the progeny of Neu5Gc-containing hESC would be permanently contaminated by Neu5Gc, hESCs were differentiated into embryoid bodies (EBs) and further into stage III spontaneously differentiated cells. EBs were cultured in the same medium as hESCs (without bFGF) in the absence of feeder cells and further differentiated cells on gelatin-coated dishes in media supplemented with ITS and fibronectin. The Neu5Gc content was found to decrease during differentiation into EB, and no Neu5Gc was detected in stage III differentiated cells. In the present analyses, Neu5Gc-containing N-glycan signals corresponding to 0.5% of total sialylated N-glycans could be reliably detected. The findings suggested that at least the majority of Neu5Gc contamination in hESC was reversible and that Neu5Gc could be gradually removed from hESC progeny in cell culture. Furthermore, the results suggested that hESC differentiation diminished the susceptibility of the cells to Neu5Gc contamination from the culture medium.
To determine whether similar decontamination as observed in hESCs also occurs in MSCs, bone marrow-derived MSCs were induced to undergo osteogenic differentiation in the presence of FBS. It was found that the differentiated cells had on average approximately 50% lower Neu5Gc contents than the original MSCs, and in one out of four experiments, N-glycan structures containing Neu5Gc could not be detected after cell culture in differentiation medium (data not shown). The results suggested that also MSC progeny could be at least partially decontaminated with respect to Neu5Gc.
Disappearance of Neu5Gc from Mesenchymal Stem Cells Cultured in Human Serum
To investigate whether MSC lines contaminated with Neu5Gc can be decontaminated by culturing them without contact to animal materials, MSCs previously cultured with FBS were moved to the same media supplemented with heat-inactivated blood group AB Rh-negative human serum instead of FBS. In immunohistochemistry, the number of Neu5Gc-positive cells significantly decreased, but after 2 weeks of culture, individual Neu5Gc-positive cells were still occasionally detected (Fig. 3). This suggested that although most cells could be easily decontaminated with regard to Neu5Gc, absolute decontamination may be difficult to reach. Mass spectrometric analysis of the Neu5Gc content in MSCs transferred from FBS to human serum containing cell culture medium indicated that Neu5Gc contamination was significantly decreased in MSCs grown in human serum (Fig. 1G).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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In general, based on the current data, it is clear that all stem cell culture conditions should be controlled with respect to Neu5Gc contamination. There are reliable biochemical and immunochemical methods for analyzing Neu5Gc content in biological materials [6, 16], and in the present study, we further demonstrated novel mass spectrometric and monoclonal antibody-based methods for controlling Neu5Gc contamination in stem cells. Given that Neu5Gc content varies between different animal proteins, it seems theoretically possible to rationally design serum replacement supplements with low and controlled Neu5Gc content, even using animal materials. Ideally, however, serum replacement protein supplements would be human proteins produced in Neu5Gc-controlled conditions. Attempts to define such media have recently been published [16].
Importantly, the present data suggest that culturing both hESCs and MSCs in appropriate conditions results in removal of Neu5Gc from stem cells or the stem cell progeny. Accordingly, recent studies have demonstrated by direct chemical analyses that hESCs lose the Neu5Gc contamination [16] and show reduced susceptibility to Neu5Gc-specific immune response [6] during cell culture with human-derived supplements. This is in accordance with our finding that most MSCs became decontaminated after prolonged culture in media in which FBS was replaced by human serum. However, our results suggested that complete decontamination may be difficult to achieve by changing culture conditions since individual cells may express Neu5Gc even after long periods. For the generation of pure Neu5Gc-negative cell populations, a negative selection procedure using for example Neu5Gc-recognizing antibodies might prove feasible.
The molecular mechanism of Neu5Gc loss from stem cells remains to be clarified [23, 24]. It is known that cells are able to secrete sialic acid-containing glycoprotein materials into culture medium, suggesting that Neu5Gc-containing material can also be secreted. Another direct mechanism of Neu5Gc loss is dilution of the molecule in stem cell progeny during normal cell growth and division. Intriguingly, the present analyses showed significant reduction in Neu5Gc levels in both hESCs and MSCs during cell culture, especially after stem cell differentiation in differentiation-inducing culture conditions.
Finally, with respect to the existing stem cells potentially contaminated with Neu5Gc, it should be noted that even normal human tissues may acquire minor amounts of Neu5Gc from dietary and other sources [24, 25]. Although it remains possible that such tissues may face low-intensity immune attack, we feel that it is unlikely that the vast majority of currently existing hESC and MSC lines that have been inadvertently exposed to Neu5Gc will be permanently contaminated and unfit for future applications if they are cultured in conditions avoiding renewed exposure to animal materials.
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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2b and interferon-14c produced by Sendai-virus-induced human peripheral blood leukocytes. Eur J Biochem 1998;253:485–493.[Medline]This article has been cited by other articles:
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  F. Mannello and G. A. Tonti Concise Review: No Breakthroughs for Human Mesenchymal and Embryonic Stem Cell Culture: Conditioned Medium, Feeder Layer, or Feeder-Free; Medium with Fetal Calf Serum, Human Serum, or Enriched Plasma; Serum-Free, Serum Replacement Nonconditioned Medium, or Ad Hoc Formula? All That Glitters Is Not Gold! Stem Cells, July 1, 2007; 25(7): 1603 - 1609. [Abstract] [Full Text] [PDF]
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